DISC1 Discord—Would the Real Disrupted in Schizophrenia Please Stand Up

10 March 2006. Since schizophrenia and related psychiatric disorders were found to strongly segregate with a familial mutation that causes a break in the gene disrupted in schizophrenia 1 (DISC1), researchers have raced to see if more subtle DISC1 variations, such as single nucleotide polymorphisms (SNPs), might associate with mental illness in the general population. While some studies support this idea, the evidence is still considered inconclusive. Now, in an article in press, published online March 1 at Human Molecular Genetics, researchers report that DISC1 polymorphisms may actually have more of an impact on expression of the genes for NUDEL, Fez1, and Lis1. The finding suggests that these DISC1 binding partners may be more disrupted in schizophrenia than DISC1 itself.

Barbara Lipska and colleagues at the National Institutes of Health, Bethesda, Maryland, together with collaborators at Johns Hopkins University School of Medicine in Baltimore and Astellas Pharma Inc., in Tsukuba, Japan, set out to determine if brain levels of the DISC1 protein are altered in people with schizophrenia, especially those with polymorphisms that have been linked to disease. Lipska and colleagues tested postmortem brain tissue samples from 79 normal individuals and 43 patients. The results were a little surprising.

The researchers found that though expression of the DISC1 gene in the brain varies with age—right after birth expression spikes, but by teenage years it falls to below neonatal values and continues to decline thereafter—there is no difference between transcript levels in normal and schizophrenia samples. The authors measured DISC1 mRNA in both the hippocampus and the dorsolateral prefrontal cortex (DLPFC) areas of the brain that have been shown to be affected by the disease. Furthermore, when the authors genotyped the samples, they found that three SNPs (hCV219779, rs821597, and rs821616) they previously identified as being associated with schizophrenia (see Callicott et al., 2005) had no effect on DISC1 transcript levels either.

But despite the normal DISC1 transcript levels, the researchers did find that DISC1 protein levels are modestly elevated (~20 percent) in hippocampal samples from schizophrenia patients compared to controls, suggesting that the protein may be more stable. But surprisingly they also found that SNP rs821597, which lies in intron 10 and therefore does not affect the protein sequence, was associated with even higher levels of DISC1 protein in the schizophrenia samples. Why this might be is unclear. Elevated protein is either due to increased production or decreased turnover. Though the SNP could conceivably affect mRNA splicing, leading to an increase in some specific transcripts and protein isoforms, the latter would not be detected by the antibody the authors used, which was raised to the C-terminal part of the protein that is absent from all the known alternatively spliced isoforms. Also, given that total DISC1 transcript levels are not affected by the SNP, decreased protein degradation/removal seems a likely explanation for the observed increase in DISC1. But how an intronic SNP might affect protein turnover is a mystery.

What is even more mysterious is that SNP rs821597 affects expression of NUDEL, Fez1, and Lis1—three proteins that interact with DISC1 (see SRF related news story and SRF news story). When the authors quantitated mRNAs in the hippocampus, they found all three were significantly lower in schizophrenia, and they also correlated with the rs821597 genotype. In tissue from schizophrenia patients homozygous for the G allele of this SNP, levels of the three transcripts were lower still (about twofold) than in tissue with the G/A or A/A genotypes—Fez1 mRNA was also significantly lower (about threefold) in the DLPFC. Curiously, the rs821597 genotype had no effect on transcript levels in normal brain tissue.

How this intronic SNP affects DISC1 protein without affecting DISC1 expression, and also affects levels of NUDEL, Fez1, and Lis1 in schizophrenia patients only, is uncertain. But the authors report that the results remain statistically significant even after the data is normalized to housekeeping proteins and transcripts, such as β-glucuronidase. They also caution, however, that the use of postmortem samples is an obvious limitation, and they note that another study using the same DISC1 antibody failed to detect any increased DISC1 in schizophrenia patients (see Sawamura et al., 2005).—Tom Fagan.

The two latest additions to the burgeoning DISC1 literature provide additional support for a role of this gene in cognitive function and schizophrenia, and suggest that more comprehensive studies will be useful as we move to a greater understanding of its role in CNS function. Koike et al. (2006) found that a relatively common mouse strain has a naturally occurring mutation in DISC1 resulting in a truncated form of the protein, similar in size (exon 7 vs. exon 8 disruptions) to that observed in the members of the Scottish pedigree in which the translocation was first detected. C57/BL/6J mice, into which mutant alleles were transferred, displayed significant impairments on a spatial working memory task similar to one used in humans (Lencz et al., 2003). These data are similar to those observed by our group (Burdick et al., 2005) and others (Callicott et al., 2005; Hennah et al., 2005; Cannon et al., 2005), although no study to date has utilized the same neurocognitive tasks. Lipska et al. (2006) report that genes and proteins (NUDEL, FEZ1) known to interact with DISC1 are also aberrant in schizophrenia postmortem tissue, with some evidence that DISC1 risk polymorphisms also influence expression across the pathway.

Taken together, these two papers suggest that the assessment of genes involved in the DISC1 pathway may be worthwhile in the evaluation of working memory function. To date, most studies have focused on risk alleles within DISC1, with little attention paid to the critical interacting genes. Studies are now underway assessing the relationship between FEZ1 and NUDEL and risk for schizophrenia in a number of populations, as well as studies examining their role in neurocognitive and neuroimaging parameters. Clearly, as the Lipska paper indicates, studies that attempt to assess multiple genes in this pathway will be critical, although the common concern of power in assessing gene-gene interactions, especially across multiple genes, may be a limitation. Moreover, these studies indicate that interaction studies will need to consider additional phenotypes other than diagnosis, and perhaps “purer” tasks of neurocognitive function may be worthwhile, as suggested by Koike et al. Finally, both of these papers underscore the fact that the next wave of genetic studies of schizophrenia will encompass the use of multiple probes, whether with neurocognitive assessments, postmortem analyses, or animal models of disease, amongst others, to fully validate the relationships between putative risk genes and the pathophysiology of schizophrenia and related disorders.

The relationship between DISC1 and neuropsychiatric disorders, including schizophrenia, schizoaffective disorder, and bipolar disorder, has now been observed in several studies. Moreover, a number of studies have demonstrated that DISC1 appears to impact neurocognitive function. Nevertheless, the molecular mechanisms by which DISC1 could contribute to impaired CNS function are unclear, and these two papers shed light on this critical issue.

Millar et al. (2005) have followed the same strategy that they so successfully utilized in their initial DISC1 studies, identifying a translocation that associated with a psychotic illness. In contrast to DISC1, in which a pedigree was identified with a number of translocation carriers, this manuscript is based upon the identification of a single translocation carrier, who appears to manifest classic signs of schizophrenia, without evidence of mood dysregulation. Two genes are disrupted by this translocation: cadherin 8 and phosphodiesterase 4B (PDE4B). The researchers' elegant set of experiments provides compelling biological evidence that PDE4B interacts with DISC1 and suggests a mechanism mediated by cAMP for DISC1/PDE4B effects on basic molecular processes underlying learning, memory, and perhaps psychosis. It remains possible that PDE4B (and DISC1) are proteins fundamentally involved in cognitive processes, and that the observed relationship to psychotic illnesses represents a final common pathway of neurocognitive impairment. This would be consistent with data from our group (Lencz et al., in press) demonstrating that verbal memory impairment specifically predicts onset of psychosis in at-risk subjects. Similarly, Burdick et al. (2005) found that our DISC1 risk genotypes (Hodgkinson et al., 2004) were associated with impaired verbal working memory. Finally, Callicott et al. (2005) found that a DISC1 risk SNP, Ser704Cys, predicted hippocampal dysfunction, an SNP which we (DeRosse et al., unpublished data) have also found to link with the primary psychotic symptoms (persecutory delusions) manifested by the patient in the Millar et al. study. This body of evidence supports the notion that these proteins play fundamental roles in the key clinical manifestations of schizophrenia.

Kamiya et al. (2005) provide another potential mechanism for these effects, suggesting that a DISC1 mutation may disrupt cerebral cortical development, hinting that studies examining the role of DISC1 genotypes on brain structure and function in the at-risk schizophrenia pediatric patients may be fruitful.

Taken together, these papers add considerable new data suggesting that DISC1 plays a key role in the etiology of schizophrenia, and places DISC1 at the forefront of the rapidly growing body of schizophrenia candidate genes.

This study describes an interesting genetic link between PDE4B (phosphodiesterase 4B) and schizophrenia that may be related to a physical interaction with DISC1 (disrupted in schizophrenia 1), another gene associated with the psychiatric disorder. The study is highly suggestive of a role for the PDE4B/DISC1 complex in schizophrenia. However, the mechanistic model suggested by the authors whereby DISC1 sequesters PDE4B in an inactive state seems overly speculative, given the results presented in this paper and in prior studies that have examined the regulation of PDE4B by phosphorylation in the absence of DISC1.

I found the paper by Kholmanskikh and colleagues, which proposes a novel
role for LIS1 in neuronal motility by bridging calcium signaling to Cdc42,
of great interest for schizophrenia research. LIS1 was originally
identified as the causative gene for lissencephaly, but cascades that
include LIS1 may have implications for schizophrenia. Several groups,
including ours, have reported that a candidate gene product for
schizophrenia, DISC1, forms a protein complex with LIS1 (Brandon et al., 2004; Kamiya et al., 2005).

My collaborators, Brian Kirkpatrick and Rosy Roberts, have observed
and presented data that DISC1 immunoreactivity is enriched in some (but not
all) of the postsynaptic densities, where Rho-family GTPases, such as
Cdc42, also occur and regulate synaptic functions (Society for Neuroscience
Meeting, 2004). Many of us agree that schizophrenia is, at least in part, a
disorder of synapses. Taken all together, it may be useful to have a
working hypothesis that a candidate susceptibility gene product for
schizophrenia, DISC1, may have an additional role in regulating synaptic
functions via Rho-family GTPases, probably in some association with LIS1.

This paper may attract researchers on schizophrenia in another context. The
authors used D-serine as a trigger of calcium signaling via activation of the NMDA receptor. Although several genes coding for proteins that are
involved in synthesis and degradation of D-serine have been associated with
schizophrenia, pathophysiological roles for D-serine remain to be
elucidated. In this sense, the impact of D-serine in activation of Cdc42
and synaptic morphology may have implications for schizophrenia
research. Personally speaking, it is the most interesting point in this
paper that the authors use D-serine in this type of experiment.

Many linkage analyses have reproducibly reported 8p21-22 as a linkage hot locus for schizophrenia. The gene coding for neuregulin-1 is regarded as a factor that contributes to the linkage peak, but other genes may also be involved. Dr. Gurling and colleagues have conducted an excellent association study and obtained evidence that the gene coding for pericentriolar material 1 (PCM1) is associated with schizophrenia.

The results from the genetic portions of this are consistent with our unpublished biological study. (The abstract of Kamiya et al. has been submitted to SFN meeting at Atlanta in October 2006.) In exploring protein interactors of disrupted-in-schizophrenia-1 (DISC1), a promising risk factor for schizophrenia and bipolar disorder, we already came across PCM1 as a potential protein interactor of DISC1. This interaction has been confirmed by yeast two-hybrid and biochemical methods. In immunofluorescent cell staining, a pool of DISC1 and PCM1 are co-stained at the centrosome. Therefore, this genetic study is really encouraging us to move beyond our preliminary study on DISC1 and PCM1.

Of interest, Gurling and colleagues reported in the paper that the cases with the PCM1 genetic susceptibility showed a significant relative reduction in the volume of orbitofrontal cortex gray matter in comparison with patients with non-PCM1-associated schizophrenia, who showed gray matter volume reduction in the temporal pole, hippocampus, and inferior temporal cortex. This may be in accordance with our previous publication (Sawamura et al., 2005) reporting the alteration in subcellular distribution of DISC1 in the orbitofrontal cortex of the patients with schizophrenia.

Although a possible link of DISC1 and PCM1 in the pathophysiology of schizophrenia is still hypothetical, the intriguing work by Dr. Gurling and colleagues may now open a window in studying the centrosomal “pathway” in association with schizophrenia. Epistatic interactions on DISC1, PCM1, and related molecules may also be of interest for future studies.

Regarding the possibility that PCM1 may have ties to DISC1, it's of interest that when PCM1 function is inhibited there is reduced targeting of centrin, pericentrin and ninein to the centrosome (1). Miyoshi and colleagues (2) report that their data indicate that DISC1 localizes to the centrosome by binding to kendrin/pericentrinB. Might there be a failure of DISC1 to localize in the centrosome in PCM1 deficiency?

Do these families with PCM1-associated schizophrenia also have a history of scleroderma? It is also of interest that PCM1 is an autoantigen target in scleroderma (3), and there is a report of cerebral involvement of scleroderma presenting as schizophrenia-like psychosis (4).

Abelson Helper Integration Site 1 (AHI1) gene is a candidate gene for schizophrenia and mutations in AHI1 underlie the autosomal recessive Joubert Syndrome in which cerebellar vermis hypoplasia is reported.(5) Increased cerebellar vermis white-matter volume has recently been reported in males with schizophrenia.(6)

It's interesting that mutations in the centrosomal protein nephrocystin-6 may also cause Joubert syndrome and that it activates ATF4. (7) Morris and colleagues (8) find that DISC1 interacts with ATF4 - a schizophrenia locus on 22q13 and ATF5. Perhaps failure of DISC1 to localize to the centrosome due to PCMI deficiency may also result in reduced activation of ATF4/5.

In view of the study by Al Sarraj and colleagues (9) finding that ATF4/5 stimulate asparagine synthetase activity might we suspect that reduced activation of ATF4 and ATF5 in schizophrenia may explain the decreased CSF asparagine levels reported (10) Perhaps asparagine synthetase might be a suitable drug target in schizophrenia. Of further relevance is the processed pseudogene for asparagine synthetase found upstream of GNAL -18p11, a region linked to bipolar disorder and schizophrenia. (11) Hirotsune and colleagues (12) report that an expressed pseudogene regulates messenger-RNA stability of its homologous coding gene.

Might we also suspect a role for DISC1 in oligodendrocyte dysfunction in schizophrenia? Reduced myelination is reported in neonatal rats deprived of asparagine?(13) It would seem relevant however that Mason and colleagues (14) find that ATF5 regulates proliferation and differentiation of oligodendrocytes, with loss of function resulting in accelerated oligodendrocyte differentiation

Den Hollander and colleagues (1) report that mutations in CEP290-nephrocystin-6 are a frequent cause of Leber's Congenital Amaurosis (LCA). Autistic signs are reported in both Joubert syndrome and LCA (2,3). Perhaps asparagine may be useful for those with LCA and dysmyelination.

The asparagine synthetase gene has been mapped to 7q21.3 (1). Childhood-onset schizophrenia/autistic disorder has been described in a child with a translocation breakpoint at 7q21. Of further interest is that alcohol/drug abuse, severe impulsivity, paranoid personality, and language delay have been reported in other family members carrying this translocation.

Maybe the increased risk of schizophrenia following famine may be explained by the fact that starvation induces expression of ATF4 and asparagine synthetase. Is there an increased risk of mutation in these genes as a long-term response to famine?

This is a useful model from Pletnikov, Ross, and colleagues, but like all models, it has some limitations. Since DISC1 is known to have a strong role in development and physiology, the development of inducible mutants is necessary to separate the two.

In the TeT-off system used in the paper, mice must be treated with doxycycline for their entire lives to keep the expression of this gene off. Doxycycline must be used at high levels and may have side effects when used this long. The TeT-on system is better because doxycycline is only used transiently for 1 week for maximum induction then washed away. The TeT-on system is also available for the same promoter used in the paper, that of the CaMKII gene.

The phenotype of reduced neurite length was obtained from in vitro neuron cultures, which are prone to artifacts. There are ways of labeling these neurons in vivo for measuring neurite length and spines. The brain phenotype was obtained by MRI. There are ways, such as adding manganese, of enhancing active pathways. This has been done in the bird brain to map song pathways.

The behavioral phenotype was similar to the recent paper from the Sawa group (Hikida et al., 2007) in that it also analyzed a transgenic mouse expressing the same C-terminal truncation of the human DISC1 gene, using the same CaMKII promoter. An important difference in the findings was a reduction of murine DISC1 (50 percent at protein level) in the Pletnikov et al. mice but none in the Sawa group mice. This issue is important because of a recent paper in Cell by the Song group (Duan et al., 2007). In that paper, RNAi was used to reduce wild-type native murine DISC1. Individual neurons with targeted DISC1 knockdown showed accelerated neurite development, greater synapse formation and enhanced excitability. Hippocampal granule cells showed accelerated morphological integration resulting in mispositioning. Unfortunately, in the Song paper they analyzed only cells with complete or no knockdown of DISC1. Partial knockdown vectors were made that achieved 75 percent reduction at the protein level but were not analyzed. Only then would it be possible to compare these morphological data with those from Pletnikov et al., which was a 50 percent reduction. Another difference was that the Song group found that DISC1 needed to interact with Nudel. Pletnikov et al. found normal levels of Nudel in the mice but lower LIS1, which could explain the brain development phenotype.